JP2020010701A - Interleukin-4 receptor-binding fusion proteins and uses thereof - Google Patents

Abstract

To provide methods capable of inhibiting cell survival, inhibiting cell proliferation, or enhancing cell death or apoptosis of a target cell expressing an IL-4R.SOLUTION: The present invention relates to interleukin-4 receptor binding fusion proteins. More specifically, the invention provides, among others, fusion proteins that include an interleukin-4 receptor binding protein moiety joined to a pro-apoptotic Bcl-2 family member protein moiety.SELECTED DRAWING: Figure 1

Description

The present invention relates to interleukin-4 receptor binding protein fusions. More specifically, the present invention provides, in part, fusion proteins comprising an interleukin-4 or interleukin-13 protein portion linked to a pro-apoptotic Bcl-2 family member protein portion.

Interleukin-4 (IL-4) is a pleiotropic cytokine produced by activated T cells and is an IL that can also bind to interleukin-13 (IL-13).
-4 receptor (IL-4R). IL-4, like many cytokines, first binds to the high-affinity receptor chain (denoted “α”), and then its IL-4-α chain complex is labeled as “γc”. Binds two low-affinity receptor chains. Therefore, IL-4
Is the IL-4 receptor alpha (IL-4Rα), which binds with high affinity (K _D = ^{−10 −10} M). The IL-4 / IL-4Rα complex can then bind with relatively low affinity to the second component of the IL-4 receptor, γc (the “type I” receptor). In addition, the IL-4 / IL-4Rα complex is interleukin-13
It can also bind to the (IL-13) receptor α1 (IL-13Rα1) (“type II” receptor).

Different cell types express different amounts of type I and type II receptor chains. For example, IL-4R
Although α is present on most cells, γc is generally expressed on hematopoietic cells and IL-13R
α1 is generally expressed on non-hematopoietic cells. Therefore, γc is a T cell, natural killer (
NK) cells, basophils, mast cells, and most mouse B cells (most human B cells express both γc and IL-13Rα1), but IL-13Rα1
Is not so.

Some bone marrow-derived cells, including macrophages and dendritic cells, have γc and IL-13R
It expresses α1 and consequently responds to both IL-4 and IL-13. IL-13Rα1 is found on most non-bone marrow derived cells, including smooth muscle and epithelial cells, but has almost no γc.

Mutant (variant) IL-4 molecules with differential selectivity for type I and type II receptors have been proposed (Junttila et al. Nature Chemical Biology 8: 990-998, 2012).

A circularly permuted molecule is one in which the ends of a linear molecule (eg, a ligand) are joined, either directly or via a linker, to form a circular molecule, which is then placed at another position. To form a new linear molecule with an end different from that of the original molecule. Cyclic permutated variants of IL-4 are described, for example, in 2000
No. 6,011,002 issued to Pastan et al.

Programmed cell death, or "apoptosis," is a common phenomenon in the development of animal cells and is regulated both positively and negatively. In addition to its involvement in neuronal and lymphoid development and homeostasis of the entire cell population, apoptosis also plays an important role in a variety of diseases and injuries resulting from abnormal regulation of the apoptotic pathway. For example, abnormal activation of neuronal cell death by apoptosis is caused by Alzheimer's disease (Barinaga, Science 281).
: 1303-1304), Huntington's disease, spinal muscular atrophy, seizure-induced neuronal damage (reviewed in Rubin, British Med. Bulle., 53 (3): 617-631, 1997; and Barinaga, Science
281: 1302-1303), and has been implicated in many neurodegenerative diseases and conditions, such as transient ischemic neuronal injury (eg, spinal cord injury). Conversely, abnormal suppression of apoptosis can result in cell hyperproliferation, leading to cancer and other hyperproliferative disorders.

Apoptosis is controlled by several proteins, including members of the Bcl-2 family. Bcl-2 was one of the first proteins identified to control apoptosis (Cleary et al., Cell 47: 19-28, 1986; Tsujimoto and Cro
Natl. Acad. Sci. USA 83: 5214-5218, 1986). Since its discovery, some B
Cl-2 related proteins ("Bcl-2 family proteins" or "Bcl-2 family members") have been identified as regulators of apoptosis (White, Genes Dev).
. 10: 1-15, 1996; Yang et al., Cell 80: 285-291, 1995; Lomonosova, E. and G. Chin
nadurai, Oncogene 27, S2-S19, 2009).

Several therapeutic agents for the treatment of neurodegenerative diseases, cancer, etc., are being investigated, but show limitations that limit their use in the clinic. For example, many chemotherapeutic agents
Although acting by inducing apoptosis in proliferating neoplastic cells, their therapeutic value is limited by their degree of toxicity to normal cells. Treatment with standard apoptosis-inhibiting molecules, such as peptide-type caspase inhibitors (eg, DEVD type), has been found to be unnecessary for clinical work due to the low membrane permeability of these inhibitors.

In an attempt to selectively eliminate cancer cells, a targeted immunotoxin (e.g., a toxic molecule such as a bacterial toxin) and a molecule derived from the targeting domain, typically from an antibody, Genetic or biochemical fusions) have been proposed. For example, diphtheria toxin (DT) variants have been generated and tested for their ability to selectively kill cancer cells (Thorpe e
t al., Nature 271: 752-755, 1978; Laske et al, Nature Medicine 3: 1362-1368, 19
97). Similarly, Pseudomonas aeruginosa (Pseudomonas) exotoxin (PE) fusion proteins have been studied as potential cancer therapies (Kreitman and Pastan, Blood 90: 252-259, 1997; Shima).
mura et al. Cancer Res. 67: 9903-9912; 2007).

The present invention relates to interleukin-4 receptor binding fusion proteins. More specifically,
The present invention provides, in part, a fusion protein comprising an interleukin-4 receptor binding protein portion linked to a pro-apoptotic Bcl-2 family member protein portion;
And its use.

In one aspect, the invention provides a fusion protein comprising an interleukin-4 (IL-4) receptor binding protein and a pro-apoptotic Bcl-2 family polypeptide. In some embodiments, the IL-4 receptor binding protein comprises a cyclic permutation (cp
) May be. In some embodiments, the Bcl-2 family polypeptide comprises a Bcl-2
It may be a pro-apoptotic Bcl-2 family polypeptide comprising an H3 domain (such as Bad, Bik / Nbk, Bid, Bim / Bod, Hrk, Bak or Bax). The BH3 domain may further include a mutation that reduces phosphorylation. A pro-apoptotic Bcl-2 family polypeptide comprising a BH3 domain further comprising a mutation that reduces phosphorylation may be a Bad polypeptide. The fusion protein may be capable of inhibiting cell survival, inhibiting cell proliferation, or enhancing cell death or apoptosis of a target cell expressing IL-4R.

In some embodiments, the IL-4 receptor binding protein is a type I or type II IL-4
It may be a mutant IL-4 or IL-13 that is selective for binding to the receptor (IL-4R). A variant IL-4 selective for binding to a type II IL-4R may comprise a KFR variant or a KF variant. A variant IL-4 selective for binding to type I IL-4R is
RGA variants may be included. Mutant IL-13 may be an A11 variant or a DN variant.

In some embodiments, the fusion protein may further include a linker. The linker may have the sequence GS or be a ubiquitin or ubiquitin variant molecule. The fusion protein may include the sequences shown in SEQ ID NOs: 24-27.

In some aspects, there is provided a nucleic acid molecule encoding a fusion protein as described herein, or a vector comprising the nucleic acid molecule, or a host cell comprising the vector. In some embodiments, as shown in SEQ ID NOs: 24-27 or SEQ ID NOs: 35-38.
A nucleic acid molecule encoding a fusion protein comprising:

In some aspects, there is provided a pharmaceutical composition comprising a fusion protein as described herein, a nucleic acid molecule encoding the fusion protein, or a vector comprising the nucleic acid molecule, or a host cell comprising the vector. You.

In some aspects, the method of inducing cell death comprises pro-apoptotic Bcl-2.
Inducing cell death by administering a fusion protein containing a family polypeptide, a nucleic acid molecule encoding the fusion protein, or a vector containing the nucleic acid molecule, or a host cell containing the vector to a subject in need thereof A method is provided.

In some aspects, the target cell expressing IL-4R is a pro-apoptotic Bcl-
Methods are provided for inducing cell death by contacting a fusion protein comprising a two family polypeptide, a nucleic acid molecule encoding the fusion protein, or a vector comprising the nucleic acid molecule.

In some embodiments, a fusion protein comprising a pro-apoptotic Bcl-2 family polypeptide, a nucleic acid molecule encoding the fusion protein, or a vector comprising the nucleic acid molecule, or a host cell comprising the vector, is required. A method for treating cancer by administering to a subject having the disease is provided.

In some aspects, the neoplastic cell expressing IL-4R is converted to a pro-apoptotic Bc
Methods for treating cancer by contacting with a fusion protein comprising a 1-2 family polypeptide, a nucleic acid molecule encoding the fusion protein, or a vector comprising the nucleic acid molecule are provided.

In some embodiments, a non-malignant cell that expresses IL-4R in a tumor microenvironment in a subject in need thereof is transformed into a fusion protein comprising a pro-apoptotic Bcl-2 family polypeptide, a nucleic acid molecule encoding the fusion protein Or a method of treating cancer by contacting with a vector comprising the nucleic acid molecule. In some embodiments, the non-malignant cells are contacted before the subject starts treatment.

In some aspects, a method of treating a hyperproliferative or differentiation disorder, comprising a fusion protein comprising a pro-apoptotic Bcl-2 family polypeptide, a nucleic acid molecule encoding the fusion protein, or comprising the nucleic acid molecule Methods are provided for treating a vector by administering to a subject in need thereof. The hyperproliferative disorder or differentiation disorder may be fibrosis or hyperplasia, an inflammatory condition or an autoimmune condition. The fibrosis or hyperplasia may be pulmonary fibrosis or hyperplasia (such as benign prostatic hyperplasia), cardiac fibrosis, or hepatic fibrosis, and the inflammatory condition is prostatitis, spring keratoconjunctivitis, atherosclerosis (Ar
the autoimmune condition may be Graves' disease.

In some aspects, the pro-apoptotic Bcl-2 family poly in a subject in need thereof for inducing cell death, or treating cancer, or treating a hyperproliferative or differentiation disorder. Use of a fusion protein comprising a peptide, a nucleic acid molecule encoding the fusion protein, or a vector comprising the nucleic acid molecule is provided.

  In various embodiments of other possible aspects, the subject may be a human.

  This summary does not necessarily describe all features of the invention.

These and other features of the present invention will become more apparent from the following description with reference to the accompanying drawings.

FIG. 1 is a diagram showing cpIL-4BAD (cpIL-4g: s-BADaa) in the pET 24a expression vector. FIG. 2 is a graph showing the effect of cpIL-4BAD (cpIL-4BADaa) on the survival rate of IL-4Rα-positive tumor cells (U251). FIG. 3 is a graph showing the effect of cpIL-4BAD (cpIL-4BADaa) (squares) on the survival of IL-4Rα-positive tumor cells (U251) in the presence of excess IL-4 (triangles). FIG. 4 is a graph showing the effect of cpIL-4BAD (cpIL-4BADaa) on the survival rate of IL-4Rα-positive tumor cells (Daudi cells). FIG. 5 is a graph showing the effect of cpIL-4BAD (cpIL-4BADaa) (triangles) on the viability of IL-4Rα-positive tumor cells Daudi in the presence of excess IL-4 (squares). FIG. 6 is a graph showing the effect of cpIL-4BAD (cpIL-4BADaa) on the number of colonies of IL-4Rα-positive tumor cells (U251). FIG. 7 shows the effect of intratumoral (square) and intraperitoneal (triangle) injection of cpIL-4BAD fusion protein in athymic mice after the development of subcutaneous glioma tumors using U251 tumor cells (circles = control). FIG. FIG. 8 is a graph showing the survival of mice treated with cpIL-4BAD (intratumor injection group = open diamond shape, intraperitoneal injection group = triangle, control = solid diamond shape). FIG. 9 is a diagram showing a pGW07 E. coli expression vector. FIGS. 10A-F show the nucleic acid (SEQ ID NOs: 29, 30, and 32) and amino acid sequences of the IL-4BAD (AB), cpIL-4BAD (CD) and cpS4-BAD (EF) fusion constructs. FIG. 21 shows SEQ ID NOs: 18, 19 and 21). FIGS. 11A-B show the nucleic acid (A; SEQ ID NO: 37) and amino acid sequence (B; SEQ ID NO: 26) of the pKFR4-BAD-H6 fusion construct.

The present disclosure provides, in part, fusion proteins comprising an IL-4R binding protein linked to a pro-apoptotic Bcl-2 family protein, and uses thereof.

  IL-4R binding protein

  IL-4R binding proteins include IL-4 and IL-13.

IL-4 protein or IL-4 "protein portion" encompasses native IL-4 protein as well as mutant IL-4 protein. A "native" or "wild-type" IL-4 sequence, as used herein, refers to a human IL-4 sequence, whether purified from a natural source or produced using recombinant techniques, as follows: (With an additional methionine at the N-terminus).

Other human IL-4 sequences that may be employed include the following amino acid sequences (with an additional methionine at the N-terminus):

In some embodiments, ILs that can be used in the fusion proteins of the present disclosure
-4 protein has enhanced selectivity for γc (type I receptor) over IL-13Rα1 (type II receptor), as described, for example, in Juntila et al. (Nature Chemical Biology 8: 990-998, 2012). Mutant IL-4 protein, or vice versa. In some embodiments, the mutant IL-4 protein with enhanced selectivity for γc (type I receptor) is described in, for example, Juntila et al. (Nature Chemical Biology 8: 990-998, 2012). The sequence of native human IL-4 (eg, SEQ ID NO: 1) or an otherwise employable IL-4 sequence (
For example, the following mutation to SEQ ID NO: 2 (numbering excludes the N-terminal methionine):
R121Q / Y124W / S125F (“RGA” or “super-4” or “S4”
(Mutant form).

In some embodiments, the variant IL-4 protein with enhanced selectivity for IL-13Rα1 (type II receptor) is a native human IL-4 (eg, SEQ ID NO: 1) sequence or otherwise employed. The following mutations to the possible IL-4 sequence (eg, SEQ ID NO: 2) (numbering excludes the N-terminal methionine): R121K / Y124F / S125R ("KFR" or "KFR4" mutant) or R121K / Y124F ( IL-4 containing "KF" variant)
It is a protein.

In some embodiments, ILs that can be used in the fusion proteins of the present disclosure
-4 protein is disclosed, for example, in U.S. Patent No. 6, issued January 4, 2000 to Pastan et al.
As described in No. 011,002, cyclic permutation is carried out (cp). In some embodiments, the cpIL-4 protein that can be used in the fusion proteins of the present disclosure is native human IL-4 (eg, SEQ ID NO: 1) or an otherwise employable IL-4 sequence (eg, SEQ ID NO: 1). Residues 38-129 of 2) (numbering excludes the N-terminal methionine) are as follows: IL-4 linked to residues 1-37 with a GGGGG linker and the first methionine residue
Contains protein.

In another embodiment that can be employed, the cpIL-4 protein that can be used in the fusion proteins of the present disclosure is a native human IL associated with an “RGA” or “super-4” or “S4” variant. -4 (eg, SEQ ID NO: 1) or residues 38-129 of the otherwise employable IL-4 sequence (eg, SEQ ID NO: 2) (numbering excludes the N-terminal methionine)
IL linked to GGGGG linker and first methionine residue at residues 1-37 as follows
-4 protein.

In other embodiments that may be employed, the cpIL-4 protein that may be used in the fusion proteins of the present disclosure may be native human IL-4 (eg, SEQ ID NO: 1) or other, in conjunction with the “KFR” variant. Residues 38 to 129 of the IL-4 sequence (eg, SEQ ID NO: 2)
(Numbering excludes the N-terminal methionine) but includes ILGG protein linked at residues 1-37 with a GGGGG linker and the first methionine residue as follows.

In other possible embodiments, the cpIL-4 protein that can be used in the fusion proteins of the present disclosure is native human IL-4 (eg, SEQ ID NO: 1) or other Residues 38 to 129 of the IL-4 sequence (e.g.
(The numbering excludes the N-terminal methionine) but includes the GGGGG linker at residues 1-37 and the IL-4 protein linked by the first methionine residue as follows.

In other alternative embodiments, the cpIL-4 protein that can be used in the fusion proteins of the present disclosure is native human IL-4 (eg, SEQ ID NO: 1) or another alternative IL-4 sequence (eg, Residues 105 to 129 of SEQ ID NO: 2 (numbering excludes the N-terminal methionine) are described, for example, in US Pat.
No. 1,011,002, comprising IL-4 protein linked at residues 1-104 with a GGGGG linker and a first methionine residue.

Examples of IL-4 proteins that can be used in the fusion proteins of the present disclosure are those described herein, as well as retaining the ability of the variant IL-4 protein to bind to the IL-4 receptor. Or, for example, Juntila et al. (Nature Chemical Biology 8:99
0-998, 2012), compared to IL-13Rα1 (type II receptor).
Native IL-4 ("mutant IL-4 protein") by at least 80%, as long as it retains the enhanced selectivity for the type receptor) or vice versa, or retains the desired biological activity.
Sequence identity (at least 85%, at least 90%, at least 95%, at least 9
8% or even at least 99% sequence identity).

An IL-4 protein of the present disclosure is a fragment that can be smaller than the 129 amino acid native IL-4 protein, wherein the IL-4 protein fragment retains the ability to bind to the IL-4 receptor. Or, for example, Junttila et al. (Nature Chemical Biology 8: 990-998,
2012) as described in IL-13Rα1 (type II receptor), retains enhanced selectivity for γc (type I receptor) or vice versa, or retains the desired biological activity And is included as a fragment of the native sequence or as a cp form or fragment thereof.

Included in this disclosure is a nucleic acid molecule encoding an IL-4 protein as described herein or known in the art, wherein the DN described herein is a DNA molecule.
It should also be understood that it includes, but is not limited to, the RNA sequence corresponding to the A sequence.

  Examples of IL-4 nucleic acid molecules include:

IL-13 protein or IL-13 "protein portion" encompasses native IL-13 protein as well as mutant IL-13 protein. "Native" or "wild type" I
The L-13 sequence, as used herein, refers to the human IL-13 sequence, whether purified from natural sources or produced using recombinant techniques, and has the following amino acid sequence (N
(With additional methionine at the end).

In some embodiments, ILs that can be used in the fusion proteins of the present disclosure
The -13 protein is a mutant IL-13 protein having enhanced selectivity for IL-13Rα1 (type II receptor) as compared to wild-type IL-13 protein. For example, I
The L-13 variant sequence has the following amino acid sequence (with additional methionine at the N-terminus):
May be included.

In some embodiments, the IL-13Rα1 (I
The variant IL-13 protein with enhanced selectivity for the type I receptor) has the following mutations relative to the sequence of native human IL-13 (SEQ ID NO: 7): L10V / E12A / V18I
/ R65D / D87S / T88S / L101F / K104R / K105T ("DN" mutant) (numbering excludes N-terminal methionine).
For example, an IL-13 variant sequence may include the following amino acid sequence (with an additional methionine at the N-terminus):

In some embodiments, ILs that can be used in the fusion proteins of the present disclosure
The -13 protein was permuted cyclically (cp). In some embodiments, the mutant cpIL-13 protein that can be used in the fusion proteins of the present disclosure is a peptide in which residues 44-114 of native human IL-13 (SEQ ID NO: 7) are as follows: 1-43 include a linker and an IL-13 protein linked by a first methionine residue.

In some embodiments, variant cpIL-13 proteins that can be used in the fusion proteins of the present disclosure are:

In another embodiment, the variant cpIL-13 protein that can be used in the fusion proteins of the present disclosure is native human IL-1 in the context of the “A11” variant.
Residues 44-114 of 3 (SEQ ID NO: 7) comprise the IL-13 protein linked to residues 1-43 at the linker and the first methionine residue as follows.

In some embodiments, variant cpIL-13 proteins that can be used in the fusion proteins of the present disclosure are:

In another embodiment, the variant cpIL-13 protein that can be used in the fusion proteins of the present disclosure is native human IL-13 in the context of a “DN” variant.
Residues 44-114 of (SEQ ID NO: 7) comprise an IL-13 protein linked to residues 1-43 by a linker and a first methionine residue as follows.

In some embodiments, variant cpIL-13 proteins that can be used in the fusion proteins of the present disclosure are:

Examples of IL-13 proteins that can be used in the fusion proteins of the present disclosure include those described herein, and further, wherein the mutant IL-13 protein is IL-1
3 retains the ability to bind to the IL-3 receptor or IL-
At least 80% sequence identity with native IL-13 ("mutant IL-13 protein"), as long as it retains enhanced selectivity for 13Rα1 (type II receptor) or retains the desired biological activity Sequences having at least 85%, at least 90%, at least 95%, at least 98% or even at least 99% sequence identity.

An IL-13 protein according to the present disclosure may be a fragment that may be smaller than a native IL-13 protein of 114 amino acids, wherein the IL-13 protein fragment is an IL-13 protein.
Retains the ability to bind to the IL-13 receptor
It is to be understood that this is conditional on retaining the selectivity for -13Rα1 (type II receptor) or retaining the desired biological activity.

Included in this disclosure are nucleic acid molecules (including, but not limited to, RNA or DNA sequences) that encode an IL-13 protein as described herein or known in the art. ).

  BCL-2 family protein

Bcl-2-related proteins or polypeptides ("Bcl-2 family proteins" or "Bcl-2 family members") are involved in the regulation of apoptosis. Bcl
-2 family proteins fall into two distinct categories: those that inhibit cell death ("anti-apoptotic" Bcl-2 family proteins) and those that enhance cell death ("pro-apoptotic" Bcl-2). Family proteins). Bcl-2 family proteins share one to four conserved Bcl-2 homology (BH) domains, designated BH1, BH2, BH3, and BH4.

Pro-apoptotic Bcl-2 family proteins include Bad (eg, accession numbers: NP116784, CAG46757, or Q92934), Bik / Nbk (eg,
Accession number: CAG30276 or Q13323), Bid (for example, Accession number: CAG2)
8531 or P55957), Bim / Bod (for example, accession number: NP619527)
, Hrk (accession number: 000019), Bak, or Bax. In some embodiments, a pro-apoptotic Bcl-2 family protein that can be used in a fusion protein according to the present disclosure is mutated to prevent phosphorylation (eg, mutation at a serine residue, eg, serine residue). To alanine).

Bad (Bcl-2-related agonist of cell death) is a regulator of programmed cell death (apoptosis). Bad is possible to form the Bcl-x _L and Bcl-2 and heterodimers, as well as positively regulate cell apoptosis by reversing their death suppressor activity. The pro-apoptotic activity of Bad is controlled by its phosphorylation. Examples of Bad proteins that can be used in the fusion proteins of the present disclosure include GenB
ank accession number CAG46757; AAH01901.1 .; and CAG467
33.1, as well as those set forth in US Pat. No. 6,737,511 (sequences incorporated herein by reference) and described herein, and variants At least 80% sequence identity with such sequences (at least 85%, at least 90%, at least 90%, as long as they retain or have the enhanced biological activity of native Bad protein).
Sequences with at least 95%, at least 98% or even at least 99% sequence identity). In some embodiments, Bad proteins that can be used in fusion proteins according to the present disclosure include a serine mutation at position 112 and / or 136 that reduces phosphorylation. In some embodiments, a Bad protein that can be used in a fusion protein according to the present disclosure comprises a serine to alanine mutation at position 112 and / or 136 that reduces phosphorylation. In some embodiments, a Bad protein that can be used in a fusion protein according to the present disclosure comprises the following sequence, or a fragment thereof:

In some embodiments, a Bad protein that can be used in a fusion protein according to the present disclosure comprises a variant sequence as follows, or a fragment thereof.

Examples of Bik / Nbk protein molecules that can be used in a fusion protein according to the present disclosure include the following sequences, or fragments thereof.

Examples of Bid protein molecules that can be used in a fusion protein according to the present disclosure include the following sequences, or fragments thereof.

Examples of Bim / Bod protein molecules that can be used in a fusion protein according to the present disclosure include the following sequences.

Examples of Hrk protein molecules that can be used in a fusion protein according to the present disclosure include the following sequences.

In some embodiments, the pro-apoptotic Bcl-2 family protein comprises
comprising at least a fragment of a cl-2 family member, wherein the pro-apoptotic Bcl
-2 family proteins or fragments can inhibit cell survival, inhibit cell proliferation, or enhance cell death or apoptosis. “Inhibiting cell survival” refers to reducing the likelihood that cells at risk of cell death will survive (eg,
At least 10%, 20%, up to 30%, or even 50%, 75%, 85% or even 90% or more). By "inhibiting cell proliferation" is meant reducing cell growth or proliferation (eg, at least 10%, 20%, up to 30%, or even 50%, 75%, 85%, or even 90% or more). It is. "Enhancing cell death or apoptosis" refers to increasing the likelihood that cells at risk of cell death will undergo apoptosis, necrosis or some other type of cell death (eg, at least 10%, 20%,
Up to 30%, or even 50%, 75%, 85% or even 90% or more). Suitable assays for measuring inhibition of cell survival, inhibition of cell proliferation, or enhancement of cell death or apoptosis are described herein or are known in the art.

Included in this disclosure is a pro-apoptotic Bcl- as described herein.
Nucleic acid molecules encoding two family members (eg, RNA or DNA sequences)
It should also be understood that this includes, but is not limited to, RNA sequences corresponding to the DNA sequences described herein.

Examples of proapoptotic Bcl-2 family member nucleic acid molecules include:

  IL-4 receptor binding protein-Bcl-2 family fusion protein

“Fusion proteins” according to the present disclosure include IL-4 and IL linked to a pro-apoptotic Bcl-2 family member by any additional sequences or moieties (such as linkers) as described herein. IL-4R binding proteins, such as -13, as well as nucleic acid molecules encoding such fusion proteins. Also included are recombinant nucleic acid molecules in which the nucleic acid sequence encoding the fusion protein is operably linked to a promoter,
Vectors containing such molecules, and transgenic cells containing such molecules.

IL-4 (including cpIL-4 and IL-4 fragments and variants) is known as Bad, Bik /
Pro-apoptotic Bcl comprising a BH3 domain as exemplified by Nbk, Bid, Bim / Bod, Hrk, Bak, or Bax or a combination thereof.
-Family polypeptides, or fragments or variants thereof, as long as they retain pro-apoptotic activity. Either type of IL-4 or derivative can be used.
For example, IL-4 or a fragment of IL-4 that binds to the IL-4 receptor can be used. in addition,
A plurality of pro-apoptotic Bcl-2 family proteins or fragments or variants thereof can be linked to IL-4 or fragments or variants thereof, or a plurality of IL-4 proteins or fragments or variants thereof can be pro-apoptotic Sex Bcl-2 family proteins or fragments or variants thereof.

IL-13 (including IL-13 fragments or variants) includes, for example, Bad, Bik / Nbk,
A pro-apoptotic Bcl-2 family polypeptide comprising a BH3 domain as exemplified by Bid, Bim / Bod, or Hrk, or a combination thereof, the combination or fragment or variant thereof retains pro-apoptotic activity As long as you can connect. Either type of IL-13 or derivative can be used. For example, IL
-13 or a fragment of IL-13 that binds to the IL-13 receptor can be used. In addition, multiple pro-apoptotic Bcl-2 family proteins or fragments or variants thereof
L-13 or a fragment or variant thereof can be linked, or a plurality of IL-13 proteins or fragments or variants thereof can be linked to a pro-apoptotic Bcl-2 family protein or a fragment or variant thereof.

cpIL-4 is Bad, Bik / Nbk, Bid, Bim / Bod, Hrk, Bak
Or a pro-apoptotic Bcl-2 family polypeptide, such as one comprising a BH3 domain as exemplified by Bax or a combination thereof, or a fragment or variant thereof, as long as the pro-apoptotic activity is retained. It is. Either type of cpIL-4 or derivative can be used. In addition, a plurality of cpIL-4 proteins or fragments or variants thereof can be linked to a pro-apoptotic Bcl-2 family protein or a fragment or variant thereof, or a plurality of pro-apoptotic Bcl-2 family proteins or a variant thereof. A fragment or variant can be ligated to the cpIL-4 protein or a fragment or variant thereof.

  Table 1 shows examples of the fusion proteins.

Pro-apoptotic B of IL-4R binding proteins such as IL-4 or IL-13
The linkage or "fusion" to a cl-2 family member may be direct such that a portion of the IL-4R binding protein is directly attached to a portion of the pro-apoptotic Bcl-2 family member. For example, one end of the amino acid sequence of an IL-4R binding protein can be directly attached to the end of the amino acid sequence of a pro-apoptotic Bcl-2 family member. For example, the C-terminus of the IL-4R binding protein can be linked to the N-terminus of a pro-apoptotic Bcl-2 family member, or the C-terminus of a pro-apoptotic Bcl-2 family member can be linked to the N-terminus of an IL-4R binding protein. Can be connected. Methods for making such fusion proteins are conventional in the art, for example using recombinant molecular biology methods.

  Linker

In some embodiments, an IL-4R binding protein moiety can be linked indirectly to a pro-apoptotic Bcl-2 family member moiety by a linker.
The linker may be, for example, a simple method of simply joining the two parts, a means of spatially separating the two parts, an IL-4R binding protein or a pro-apoptotic Bcl.
-2 family members, or combinations thereof, can be useful in providing additional functionality.

In general, a linker that links the IL-4R binding protein portion and the pro-apoptotic Bcl-2 family member portion comprises (1) the ability of the two molecules to fold and act independently of each other, and (2) the functionality of the two portions. Has no tendency to produce ordered secondary structures that can interfere with the domain, (3) minimizes hydrophobic or charged properties that can interact with the functional protein domain, and / or (4) spaces between the two regions Can be designed to provide strategic separation. For example, in one example, the IL-4R binding protein is spatially separated from the pro-apoptotic Bcl-2 family member, such that the IL-4R binding protein becomes pro-apoptotic Bcl.
It may be desirable to interfere with the activity of -2 family members and / or prevent pro-apoptotic Bcl-2 family members from interfering with the activity of IL-4R binding proteins. The linker can be, for example, an IL-4R binding protein and a pro-apoptotic B
Instability in binding between cl-2 family members, enzyme cleavage sites (eg, cleavage sites for proteases), stability sequences, molecular tags, detectable labels, or various combinations thereof. It can also be used to provide. In some embodiments, the linker is an IL-4R binding protein (such as within a cp molecule) or a pro-apoptotic Bcl.
-2 family member can exist between the two domains.

The linker can be bifunctional or multifunctional, that is, at least approximately
A first reactive functional group at or near a first end of a linker capable of or modified to bind to an L-4R binding protein, and a modified pro-apoptotic Bcl A second reactive functional group at or near the opposite end of the linker capable of being attached to or modified to bind to the -2 family member.
The two or more reactive functional groups are the same (ie, the linker is homobifunctional), or
Or they can be different (ie the linker is heterobifunctional).

The length and composition of the linker can vary considerably. The length and composition of the linker will generally depend on the intended function of the linker and, optionally, ease of synthesis, stability, particular chemicals and / or
Alternatively, the selection is made in consideration of resistance to a temperature parameter and other factors such as biocompatibility. For example, the linker may be an IL-4R binding protein and / or a pro-apoptotic Bc.
It should not significantly interfere with the activity of 1-2 family members.

Suitable linkers for use in the fusion proteins according to the present disclosure include peptides.
The linker can be attached to the IL-4R binding moiety and / or the pro-apoptotic Bcl-2 family member moiety using recombinant DNA technology. Such methods are well known in the art, and details of this technology are described, for example, in Sambrook, et al. Molecular Cloning.
: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbo
r Laboratory Press, Cold Spring Harbor, NY, 1989 or Ausubel et al. Current
Protocols in Molecular Biology, found in John Wiley & Sons, 1994, or an updated version thereof.

The linker peptide has 1 to 500 amino acid residues (1 to 100, 1 to 50, 6 to 30, 1
-40, 1-20, or less than 30 amino acids or 5-10 amino acids). In some embodiments, the linker can be 2, 3, 4, 5, 6, 7 or 8 amino acids in length, or about 10, 20, 30, 40 or 50 amino acids in length. .

Typically, surface amino acids in the flexible protein region include Gly, Asn and Ser.
And such amino acids can be used in the linker sequence. Thr and A
Other neutral amino acids such as la can also be used in the linker sequence. Additional amino acids can be placed within the linker to provide unique restriction enzyme sites in the linker sequence to facilitate fusion construction. In some embodiments, the linker may comprise, for example, the amino acid sequence Gly-Ser (GS) or may comprise the amino acid sequence Gly-Ser (GS).
Or the ubiquitin sequence:
GGGSMQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIFAGRQLEDGRTLSDYNIQRESTLHLVLRLRGG
GS (SEQ ID NO: 28) or a variant thereof may be included. Ubiquitin molecules suitable for use as linkers are described, for example, in Bachran, C et al., "Anthrax toxin-mediated delivery of
the Pseudomonas exotoxin A enzymatic domain to the cytosol of tumor cells via cl
eavable ubiquitin fusions MBio. 2013 Apr 30; 4 (3): e00201-13, or PCT Publication No.W
O / 2012/139112.

In one example, a peptide linker susceptible to cleavage by enzymes of the complement system, urokinase, tissue plasminogen activator, trypsin, plasmin, or another enzyme with proteolytic activity may be used. According to another example, the IL-4R binding protein is
The attachment can be via a linker susceptible to cleavage by an enzyme having proteolytic activity such as urokinase, tissue plasminogen activator, plasmin, thrombin or trypsin. In addition, an IL-4R binding protein can be attached to a pro-apoptotic Bcl-2 family member via a disulfide bond (eg, a disulfide bond on a cysteine molecule). For example, in connection with pro-apoptotic Bcl-2 family proteins, many tumors naturally release high levels of glutathione (reducing agent), which can reduce disulfide bonds, and then provide a site for delivery. Release of the pro-apoptotic Bcl-2 family member.

In some embodiments, a fusion protein according to the present disclosure may comprise a pro-apoptotic Bcl-2 family member and an IL-4R binding protein linked by a cleavable linker region. In another embodiment, the cleavable linker region can be a protease cleavable linker, although other linkers, such as those cleavable by small molecules, may be used. Examples of protease cleavage sites include those that are cleaved by factor Xa, thrombin and collagenase. In one example, protease cleavage sites include those that are cleaved by a protease associated with the disease. In another example, a protease cleavage site is one that is cleaved by a protease that is generally associated with or upregulated by cancer. Examples of such proteases include uPA, the matrix metalloprotease (MMP) family, caspases, elastase, prostate specific antigen (PSA, serine protease), and the plasminogen activator family, as well as fibroblast activating proteins. is there. In yet another example, the cleavage site is cleaved by a protease secreted by the cancer-associated cell. Examples of these proteases include matrix metalloproteases, elastase, plasmin, thrombin, and uP
A. In another example, a protease cleavage site is one that is associated with or up-regulated by a particular cancer. The exact sequence can be obtained in the art and there should be no difficulty for a person skilled in the art to select a suitable cleavage site. As an example, the protease cleavage region targeted by Factor Xa is IEGR. The protease cleavage region targeted by enterokinase is DDDDK. The protease cleavage region targeted by thrombin is LVPRG. In one example, the cleavable linker region is one that is targeted by an intracellular protease.

The linker can be attached to the IL-4R binding protein moiety and / or the pro-apoptotic Bcl-2 family member moiety using routine techniques as are known in the art.

Preparation of IL-4R binding protein / pro-apoptotic Bcl-2 family fusion protein

Fusion proteins can be prepared using routine methods as known in the art. The fusion protein, and further modifications thereof, can be produced, for example, by designing and operating a nucleic acid encoding the fusion protein using recombinant DNA technology, or by peptide synthesis. Modifications to the fusion protein may be made, for example, by modifying the fusion protein polypeptide itself using chemical modification and / or limited proteolysis. Combinations of these methods may also be used to prepare fusion proteins.

Methods for cloning and protein expression are well known in the art, and a detailed description of techniques and systems for recombinant protein expression can be found in, for example, Current Protocols in Protein
Science (Coligan, JE, et al, Wiley & Sons, New York).
One skilled in the art will appreciate that a wide variety of expression systems can be used to provide the recombinant protein. Thus, fusion proteins are used in prokaryotic hosts (eg, E. coli,
Aeromonas salmonicida or Bacillus subtilis) or a eukaryotic host (eg,
Yeast (Saccharomyces) or Pichia; mammalian cells such as COS, NI
H3T3, CHO, BHK, 293, or HeLa cells; or insect cells (baculovirus). Fusion proteins can be purified from host cells using standard techniques known in the art.

The sequences for various examples of fusion proteins are shown in Table 1. Variants and homologues of these sequences may be obtained by standard techniques (eg, Ausubel et al, Cur.
rent Protocols in Molecular Biology, Wiley & Sons, NY (1997 and updated); Sambrook
et al., Sambrook, et al. Molecular Cloning: A Laboratory Manual.2nd ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbo
r, NY, 1989, or an updated version thereof). For example,
The nucleic acid sequence can be obtained by extracting mRNA directly from a suitable organism, such as Aeromonas hydrophila, and then PCR amplifying the gene from an mRNA template (eg, by RT-PCR) or from genomic DNA, Can be obtained by synthesizing Alternatively, the IL-4R binding moiety or the pro-apoptotic Bcl-2
Nucleic acid sequences encoding any of the family members may have the appropriate c
It can be obtained from a DNA library. The isolated cDNA is then inserted into a suitable vector, such as a cloning vector or an expression vector.

Mutations can be introduced (if desired) at specific, pre-selected locations by in vitro site-directed mutagenesis techniques well known in the art. Mutations can be introduced by deletion, insertion, substitution, inversion, or a combination of one or more of the appropriate nucleotides making up the coding sequence.

The expression vector can further include control elements, such as transcription elements, required for efficient transcription of the sequence encoding the fusion protein. Examples of control elements that can be incorporated into the vector include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Vectors containing control elements operably linked to a nucleic acid sequence encoding a genetically modified fusion protein can be used to make the fusion protein.

The expression vector may be an affinity tag (eg, a metal affinity tag, a histidine tag, an avidin /
A sequence encoding streptavidin, a sequence encoding glutathione-S-transferase (transferase) (GST), a sequence encoding maltose binding protein (MBP) or a sequence encoding biotin). Heterologous nucleic acid sequences that facilitate purification may additionally be included. In one example, such a tag can be attached to the N- or C-terminus of the fusion protein, or can be located within the fusion protein. The tag can be removed from the expressed fusion protein prior to use according to methods known in the art. Alternatively, the fusion protein can be retained if the tag does not interfere with the ability of the fusion protein to achieve the desired activity.

The fusion protein can include one or more linkers, as well as other moieties, as desired and / or as discussed herein. These include binding regions such as avidin or epitopes, or tags such as polyhistidine tags, which may be useful for purification and processing of the fusion protein, as well as other linkers as described herein. Can be included. In addition, a detectable marker can be attached to the fusion protein so that trafficking of the fusion protein in the body or cells can be easily monitored. Such markers include radionuclides, enzymes, fluorophores, chromophores, and the like.

One skilled in the art will appreciate that DNA can be altered in a number of ways without affecting the biological activity of the encoded protein. For example, PCR can be used to generate a change in the DNA sequence encoding the fusion protein. Such alterations in the DNA sequence encoding the fusion protein can be used to optimize codon preference in the host cell used to express the protein, or other changes that enhance expression. Sequence changes may be included.

Direct covalent attachment of an IL-4R binding protein to a pro-apoptotic Bcl-2 family member, or attachment via a linker, may take various forms as known in the art. For example, the covalent bond may be in the form of a disulfide bond. Ingredient 1
The DNA encoding one can be designed and manipulated to include a unique cysteine codon. The second component can be derivatized with a sulfhydryl group that is reactive with the cysteine of the first component. Alternatively, a sulfhydryl group can be introduced using solid phase polypeptide technology, either on its own or as part of a cysteine residue. For example, the introduction of a sulfhydryl group into a peptide has been reported by Hiskey (Peptides 3: 137, 1981).

Assay

Fusion proteins can be assayed using standard techniques known in the art or described herein.

For example, the ability of the fusion protein to kill cells or inhibit their growth can be assayed in vitro using a suitable cell, typically a cell line expressing a target or cancer cell. Generally, cells of the selected test cell line are grown to an appropriate density and the candidate fusion protein is added. The fusion protein can be present in the culture in at least about 1 ng / mL, at least 1 μg / mL, or at least 1 mg / mL, such as about 0.01 μg / mL to about 1 mg / mL, about 0.10 μg / mL to about 0.5 mg / mL. About 1 μg / mL to about 0.4
mg / mL can be added. In some examples, serial dilutions are examined. After an appropriate incubation time (e.g., about 48-72 hours), cell survival or growth is assessed. Methods for determining cell survival are well known in the art and include the resazurin reduction test (Fields & Lanca).
ster Am. Biotechnol. Lab., 11: 48-50, 1993; O'Brien et al, Eur. J. Biochem., 267.
: 5421-5426, 2000 or US Patent No. 5,501,959), a sulforhodamine assay (Rubinstein et al, J. Natl. Cancer Inst., 82: 113-118, 1999) or a neutral red dye test ( Kitano et al, Euro.J. Clin.Investg., 21: 53-58, 1991; West et al, J. Inv
estigative Derm., 99: 95-100, 1992) or trypan blue assay. Numerous commercially available kits may also be used, for example, CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Cytotoxicity is determined by comparing cell survival in a treated culture to cell survival in one or more control cultures, such as untreated cultures and / or control compounds (typical Include a culture pre-treated with a known therapeutic agent, or other suitable control.

Further assays are described, for example, in Crouch et al. (J. Immunol. Meth. 160, 81-8); Kangas et al.
Med. Biol. 62, 338-43, 1984); Lundin et al. (Meth. Enzymol. 133, 27-42, 1986); Pett.
(Comparison of J. Biolum. Chemilum. 10, 29-34, 1995); and Cree et al. (AntiCance
r Drugs 6: 398-404, 1995). Cell viability was determined by MTT (3- (4,5-
(Dimethylthiazolyl) -2,5-diphenyltetrazolium bromide) (Barltrop, Bioorg. & Med. Chem. Lett. 1: 611).
, 1991; Cory et al, Cancer Comm. 3, 207-12, 1991; Paull J. Heterocyclic Chem. 25
, 911, 1988). Assays for cell viability are also available commercially. These assays include CELLTITER-GLO® Luminescent Cell Viabilit, which detects ATP using luciferase technology and quantifies health or cell number in culture.
y Assay (Promega) and lactate dehydrogenase (LDH) cytotoxicity assay (Promeg
a) CellTiter-Glo® Luminescent Cell Viability Assay,
It is not limited to these.

Fusion proteins that provide selectivity for a particular type of cancer may be tested for their ability to target such particular cancer cell types. For example, IL-4R
A fusion protein containing a specific IL-4 that targets cells presenting type I or type II will differ in the ability of the fusion protein to kill cancer cells and the different types of normal or cancer cells (eg, , Which do not express the type I or type II of IL-4R) can be assessed for their ability to selectively target such cells. Alternatively, to determine whether a fusion protein comprising a type I or type II receptor-specific IL-4 can selectively target a particular type of cell, a flow site as known in the art. A metrology method may be used. Binding of the labeled antibody to the bound fusion protein will indicate binding of the fusion protein to the target.

Similarly, assays for measuring cell apoptosis are known in the art.
Apoptotic cells contain chromatin condensation, cell shrinkage and blebbing
And can be clearly observed using light microscopy. Biochemical features of apoptosis include DNA fragmentation, protein cleavage at specific locations, increased mitochondrial membrane permeability, and the appearance of phosphatidylserine on cell membrane surfaces. Assays for apoptosis are known in the art. Examples of assays include the TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) assay, the caspase activity (specifically, caspase-3) assay, and the assays for fas-ligand and annexin V. Commercially available products for detecting apoptosis include, for example, Apo-ONE® Hom
ogeneous Caspase-3 / 7 Assay, FragEL TUNEL kit (ONCOGENE RESEARCH PRODUCTS, San
Diego, Calif), ApoBrdU DNA Fragmentation Assay (BIO VISION, Mountain View, Cali)
f) and Quick Apoptotic DNA Ladder Detection Kit (BIO VISION, Mountain View, Ca
lif) is included.

A variety of cell lines suitable for testing candidate fusion proteins are known in the art and many are commercially available (eg, American Type Culture Collection (American Typ.
e Culture Collection), from Manassas, Va). Similarly, animal models are known in the art and many are commercially available.

  Therapeutic indicators and uses

IL-4R binding proteins and pro-apoptotic Bc as described herein
Fusion proteins comprising 1-2 family members can be used for various therapeutic purposes. In general, the fusion proteins described herein involve cells that express IL-4R and any disease in which inhibiting cell proliferation or enhancing cell death would be beneficial. It can also be used to treat or prevent a disorder or condition. In some embodiments, the fusion proteins described herein involve cells expressing type I or type II IL-4R, and select one type of receptor over the other. Can be used to treat or prevent any disease, disorder or condition where it would be useful to inhibit cell proliferation or enhance cell death.

In some embodiments, a fusion protein comprising a pro-apoptotic Bcl-2 family member is used to induce apoptosis or cell death, or to treat a disorder associated with abnormal apoptosis or cell proliferation, such as cancer. Can be used for
As used herein, the terms `` cancer, '' `` cancerous, '' `` hyperproliferative, '' or `` neoplastic ''
A cell that has the potential for autonomous growth (eg, an abnormal condition or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic conditions can be categorized as "pathological" (eg, deviating from normal but not associated with the condition). Thus, what is meant by "cancer" or "neoplasm" is the undesirable growth of cells that does not serve physiological functions. Generally, a neoplastic cell is one that has been released from its normal mitotic regulation, ie, its growth is not controlled by normal biochemical and physical effects in the cellular environment. In most cases, neoplastic cells proliferate to form clones of either benign or malignant cells. Examples of cancers or neoplasms include, but are not limited to, transformed and immortalized cells, tumors, and carcinomas such as breast cell carcinoma and prostate carcinoma. The term "cancer" includes cell growth that is technically benign but at risk of becoming malignant. What is meant by "malignant tumor" is the abnormal growth of any cell type or tissue. The term “malignancy” includes cell growth that is strictly benign but at risk of becoming malignant. The term includes any cancer, carcinoma, neoplasm, neoplasm, or tumor. These terms include, therefore, any type of cancerous growth or transformation process, metastatic tissue or malignant, regardless of histopathological type or invasive stage Cell, tissue or organ. In some embodiments, a fusion protein comprising a pro-apoptotic Bcl-2 family member is not used in connection with a cancer that affects stem cells.

Most cancers fall into three broad histological categories. That is, a major cancer that is epithelial cells or cells that line the outer or inner surface of an organ, gland, or other body structure (eg, skin, uterus, lung, breast, prostate, stomach, intestine). And prone to metastases; sarcomas derived from connective or supporting tissues (eg, bone, cartilage, tendons, ligaments, fat, muscle); and blood tumors derived from bone marrow and lymphoid tissues. Examples of cancer include, but are not limited to, carcinoma, sarcoma, and hematopoietic neoplastic disorders (eg, leukemia).

The carcinoma can be adenocarcinoma (commonly occurring in glands or organs capable of secretion such as the breast, lung, colon, prostate or bladder) or squamous cell carcinoma (arising from squamous epithelium and covering most areas of the body) Which generally develops in

Sarcomas include osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelial sarcoma or mesothelioma (membrane lining of body cavity) , Fibrosarcoma (fibrous tissue)
, Hemangiosarcoma or hemangioendothelioma (vascular), liposarcoma (adipose tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxoid sarcoma (primitive embryonic connective tissue, or mesenchyme) It may be a sex or mixed mesoderm tumor (mixed connective tissue type).

Hematopoietic neoplastic disorders include diseases involving hyperplastic / neoplastic cells of hematopoietic origin and arise, for example, from the bone marrow, lymphoid or erythroid lineage or progenitor cells thereof. Preferably, the disease results from poorly differentiated acute leukemia (eg, erythroblastic leukemia and acute megakaryoblastic leukemia). Examples of additional myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML), and also include lymphoid malignancies. Tumors include acute lymphoblastic leukemia (ALL) (B
Strain ALL and T strain ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia, and Waldenstrom's macroglobulinemia It will not be done.

Additional types of malignant lymphomas include non-Hodgkin's lymphoma and variants thereof, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's Disease and Reed-Stemberg
) Diseases include, but are not limited to:

Cancers can also be found in the organs from which they originated, that is, the "primary sites" such as the breast, brain,
It is sometimes named based on cancers such as lung, liver, skin, prostate, testis, bladder, colon and rectum, cervix, uterus, and the like. This nomenclature will continue to be used, even if the cancer has spread to another body site different from the primary site. Cancers named based on primary site can be associated with a histological classification. For example, lung cancer is generally small cell lung cancer or non-small cell lung cancer (which can be squamous cell carcinoma, adenocarcinoma), or large cell carcinoma, and skin cancer is generally basal cell carcinoma, squamous cell carcinoma, or melanoma. is there. Lymphomas can occur in lymph nodes associated with the head, neck and chest, as well as in abdominal lymph nodes or axillary or inguinal lymph nodes. Identification and classification of cancer types and stages can be performed, for example, by Surveillance, Epidemiology, and End Results (SEE
R) may be performed using information provided by the Program of the National Cancer Institute.

In some embodiments, the fusion protein comprising a pro-apoptotic Bcl-2 family protein member, or fragment thereof, is a gastric carcinoma, invasive pituitary adenoma, cholangiocarcinoma, cervical cancer, lymphoma, melanoma, chronic lymphocyte. Leukemia, non-Hodgkin's lymphoma, follicular lymphoma, pancreatic cancer, colorectal cancer, colon cancer, thyroid cancer, liver cancer, ovarian cancer, prostate cancer, bladder cancer, renal cell carcinoma, mesothelioma, rhabdomyosarcoma, breast cancer, It can be used to treat cancer such as non-small cell lung cancer, head and neck cancer, or Kaposi carcinoma.

In some embodiments, the fusion protein comprising a pro-apoptotic Bcl-2 family protein member, or a fragment thereof, is a glioma, meningeal tumor, diffuse endogenous glioma, medulloblastoma, neuroblastoma Anaplastic astrocytoma, glioblastoma multiforme, metastatic brain cancer, or CNS
It can be used to treat CNS cancer such as lymphoma.

The fusion proteins can be used to treat, stabilize or prevent cancer.
The fusion protein may be a relapsed and / or refractory cancer (including a cancer that has not responded to other anti-cancer treatments), including painless, local, distant, or metastatic, locally advanced, and highly invasive cancers. It can also be used in the treatment of cancer. In this context, the fusion protein can have either a cytotoxic or cytostatic effect, such as a reduction in the number or growth of cancer cells, a reduction in tumor size, a reduction in tumor size. Slowing or preventing an increase in the disease, increasing disease-free survival between the disappearance or removal of the tumor and its reappearance, preventing the first or later onset of the tumor (eg, metastasis), increasing the time to progression, associated with the tumor One or more adverse symptoms or an increase in the overall survival of a subject with cancer.

Other examples of proliferative and / or differentiative disorders that can be treated using fusion proteins comprising pro-apoptotic Bcl-2 family members include proliferative non-malignant diseases such as pulmonary fibrosis or hyperplasia (eg, benign prostate hyperplasia, etc.). ), Cardiac fibrosis, or hepatic fibrosis; inflammatory conditions such as prostatitis, keratoconjunctivitis verruca, atherosclerosis, idiopathic pneumonia; and autoimmune conditions such as Graves' disease.

In some embodiments, the fusion protein comprising a pro-apoptotic Bcl-2 family protein member, or a fragment thereof, is capable of inhibiting cell survival, inhibiting cell proliferation, or enhancing cell death or apoptosis. it can. In some embodiments, the IL-4R binding protein-pro-apoptotic Bcl-2 family fusion protein is a suitable protein, such as IL-4 alone, IL-4 linked to a non-pro-apoptotic Bcl-2 family protein, and the like. Compared to a control, it can inhibit cell survival, inhibit cell proliferation, or enhance cell death or apoptosis. Suitable controls may also include previously established standards. Thus, any test or assay for determining the activity or efficacy of an IL-4R binding protein-pro-apoptotic Bcl-2 family fusion protein may be compared to established standards, each time It is not necessary to include a control for the application. “Inhibiting cell survival” refers to reducing the likelihood that cells at risk of cell death will survive (eg, at least 10%, 20%, 3%).
0%, or even 50%, 75%, 85% or even 90% or more). “Inhibiting cell proliferation” refers to reducing cell growth or proliferation (eg, at least 10
%, Up to 20%, up to 30%, or even more than 50%, 75%, 85% or even 90%). "Enhancing cell death or apoptosis" refers to increasing the likelihood that cells at risk of cell death will undergo apoptosis, necrosis or some other type of cell death (eg, at least 10%, 20%, Up to 30% or 50%, 75
%, 85% or even 90% or more).

In some embodiments, the fusion protein comprising a pro-apoptotic Bcl-2 family protein member, or a fragment thereof, has a greater number of cells compared to cells cultured under similar conditions but without contact with said fusion protein. At least 20%, up to 30%, or 50%,
75%, 85% or even 90% or more can inhibit cell survival, inhibit cell proliferation, or enhance cell death or apoptosis. Suitable assays for measuring inhibition of cell survival, inhibition of cell proliferation, or enhancement of cell death or apoptosis include:
They are described herein or are known in the art.

In some embodiments, the fusion protein comprising a pro-apoptotic Bcl-2 family protein member, or a fragment thereof, in inhibiting cell survival, inhibiting cell proliferation, or enhancing cell death or apoptosis. IC ₅₀ is about 0.1n
g / mL to any number in the range of about 10,000 ng / mL or in between, such as about 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL,
50 ng / mL, 75 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL
mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 4
50 ng / mL, 500 ng / mL, 550 ng / mL, 600 ng / mL, 650 ng
/ ML, 700 ng / mL, 750 ng / mL, 800 ng / mL, 850 ng / mL,
Such as 900 ng / mL, 950 ng / mL, or 1000 ng / mL.

"Target cells" include neurons, lymphocytes, stem cells, epithelial cells, cancer cells, neoplastic cells,
Examples include, but are not limited to, immune cells, non-malignant cells of the tumor microenvironment, hyperproliferative cells, and the like. The target cell selected will depend on the disease or injury or condition intended to be treated with the fusion protein.

  Pharmaceutical composition, dosage and administration

Pharmaceutical compositions according to the present disclosure can include one or more fusion proteins and one or more non-toxic, pharmaceutically acceptable carriers, diluents, excipients, and / or adjuvants. Such compositions may be suitable for use in treating therapeutic indicators as described herein.

If desired, other active ingredients may be included in the composition. Thus, in some embodiments, a fusion protein comprising a pro-apoptotic Bcl-2 family member comprises:
It may be administered in a therapeutically effective amount together with one or more anti-cancer or other therapies. The fusion protein (s) can be administered before, during, or after treatment with an anti-cancer therapy or other therapy. "Anti-cancer therapy" is a compound that prevents or delays the growth and / or metastasis of cancer cells,
A composition, or a procedure (eg, surgery). Such anti-cancer therapies include surgery (eg, removal of all or part of a tumor), chemotherapeutic treatment, radiation, gene therapy, hormonal manipulation, immunotherapy (eg, therapeutic antibodies and cancer vaccines) and anti-cancer therapy. Includes but is not limited to sense or RNAi oligonucleotide therapy. Examples of useful chemotherapeutic agents include hydroxyurea, busulfan, cisplatin, carboplatin, chlorambucil, melphalan, cyclophosphamide, ifosfamide, daunorubicin,
Doxorubicin, epirubicin, mitoxantrone, vincristine, vinblastine,
Vinorelbine, etoposide, teniposide, paclitaxel, docetaxel, gemcitabine, cytosine, arabinoside, bleomycin, neocarcinostatin (neocarcinostatin)
), Suramin, Taxol, Mitomycin C, Avastin, Herceptin®
, Fluorouracil, temozolamide, and the like, but are not limited thereto. It is to be understood that the fusion protein (s) is also suitable for use with standard combination therapy using two or more chemotherapeutic agents. Anti-cancer therapies include new compounds or treatments that will be developed in the future.

Sensitizers such as the fusion protein radiosensitizers (eg, Diehn et al, J. Natl. Canc.
er Inst. 98: 1755-7, 2006). Generally, a sensitizer is any agent that increases the activity of the fusion protein. For example, a sensitizer will enhance the ability of the fusion protein to inhibit cancer cell growth or kill cancer cells. Examples of sensitizers include antibodies to IL-10, bone morphogenetic proteins and HDAC inhibitors (see, for example, Sakariassen et al, Neoplasia 9 (ll): 882-92, 2007). These sensitizers can be administered before or during treatment with the fusion protein. Examples of dosages of such sensitizers include at least 1 μg / mL, such as at least 10 μg / mL, at least 100 μg / mL, for example, 5-100 μg / mL or 10-90 μg / mL. The sensitizer can be administered daily, three times a week, twice a week, once a week, or once every two weeks. The sensitizer can also be administered after treatment with the fusion protein is completed.

The fusion protein may be used as part of a neoadjuvant therapy (as opposed to a primary therapy), as part of an adjuvant therapy regimen, if it is intended to cure the cancer in the subject. Fusion proteins can be progressive and / or invasive overgrowth (eg,
Overt disease in subjects who are not amenable to cure by local modalities such as surgery or radiation therapy), metastatic disease, locally advanced disease and / or refractory tumors (e.g., cancer or (Tumor) treatment, as well as at various stages of tumor development and progression. "Primary therapy" refers to a first-line treatment upon the first diagnosis of cancer in a subject. Examples of first line therapy include surgery, a wide range of chemotherapy and radiation therapy. "Adjuvant therapy" refers to therapy that follows the primary therapy and is applied to subjects at risk for recurrence. Adjuvant systemic therapy is initiated shortly after primary therapy, for example, 2, 3, 4, 5, or 6 weeks after the last primary therapy treatment, to delay recurrence and prolong subject survival or healing. As discussed herein,
It is contemplated that the fusion protein can be used alone or in combination with one or more other chemotherapeutic agents as part of adjuvant therapy. The combination of the fusion protein with a standard chemotherapeutic agent can act to improve the efficacy of the chemotherapeutic agent and can therefore be used to improve standard cancer treatments. The present application may be of particular interest in the treatment of drug resistant cancers that are unresponsive to standard treatments.

In cancer, the tumor microenvironment contains both malignant and non-malignant cells. The tumor microenvironment can be identified using one or more of the following criteria: (a) a region immediately adjacent to the malignant cells or containing non-malignant cells sharing the same physiological environment; (b) expanded Tumor area; (
c) areas of inflammation surrounded or in close proximity to the tumor; (d) areas of increased number or rate of proliferation of regulatory T cells; and (e) tumor-associated macrophages, dendritic cells, bone marrow-derived suppressor cells, Area where Th2 cells or fibrocytes are elevated. In the context of non-solid tumor types, the tumor microenvironment may also be determined by local cell-cell interactions between malignant cells and between malignant cells and any adjacent or neighboring non-malignant cells. Such interactions include, for example, cell adhesion events of soluble mediators produced by one cell (malignant or non-malignant) to another cell (malignant or non-malignant) within the tumor microenvironment and / or paracrine. Effects can be included.

Non-malignant cells within the tumor microenvironment may be important for tumor initiation and progression (Reynolds et al., Cancer Res., 1996, 56 (24): 5754-5757). Non-malignant cells, also called stromal cells, occupy or accumulate in the same cell space as malignant cells, or adjacent or adjacent to malignant cells, and modulate tumor cell growth or survival. For example, non-malignant cells that normally function to support inflammatory and immune responses may be able to contribute to tumor initiation or progression. Thus, in other possible embodiments, inhibiting cell survival of non-malignant cells expressing type I or type II IL-4R in a tumor microenvironment, inhibiting cell proliferation, or inhibiting cell death or apoptosis. For enhancement, a fusion protein comprising a pro-apoptotic Bcl-2 family member can be used. Such non-malignant cells include bone marrow-derived suppressor cells (eg, bone marrow-derived monocytes and tie-2 expressing monocytes) or antigen-presenting cells (eg, macrophages, dendritic cells, B cells) present in the tumor microenvironment. Inhibition of a subset of T cells (eg, regulatory T cells and Th2 helper cells), which can be immunomodulatory or inflammatory cells, such as those that support tumor progression, and / or one or more within the tumor microenvironment Can suppress the production of inflammatory cytokines. Of the non-malignant cells of the tumor microenvironment, regulatory T cells are frequently observed in many tumors, and include Hodgkin's lymphoma and non-Hodgkin's lymphoma (Shi et al,
Ai Zheng., 2004, 23 (5): 597-601 (summary only)), malignant melanoma (Viguier et al., J. Im
munol., 2004, 173 (2): 1444-53; Javia et al, J. Immunother., 2003, 26 (l): 85-93)
And the ovaries (Woo et al, Cancer Res., 2001, 61 (12): 4766-72), the gastrointestinal tract (Ichihara e
t al., Clin Cancer Res., 2003, 9 (12): 4404-4408; Sasada et a /., Cancer, 2003, 98 (
5): 1089-1099), breast (Liyanage et al, J Immunol., 2002, 169 (5): 2756-2761), lung (Woo et al, Cancer Res., 2001, 61 (12): 4766-72). ) And pancreas (Liyanage et al, J Im
munol., 2002, 169 (5): 2756-2761). Regulatory T cells are recruited at the tumor site in response to chemokines secreted by the tumor cells. For example, Curiel et al., Nat. Med.,
2004, 10: 942-949. Increased numbers of regulatory T cells can also correlate with poor prognosis (Curiel et al, Nat. Med., 2004, 10: 942-949; Sasada et al, Cancer, 2003, 98: 1).
089-1099). In contrast, regulatory T cells have been observed to decrease after chemotherapy (Beyer
et al, Blood, 2005, 106: 2018-2025). In the tumor microenvironment, compared to Th1 cells, T
The percentage of h2 cells may be high, which is associated with poor prognosis and survival. In another embodiment that can be employed, a pro-apoptotic Bcl- such as cpIL-4-Bad, according to the present invention.
Fusion proteins comprising members of the two families are useful, for example, to restore Th1 >> Th2 balance by depleting Th2 cells. In some embodiments, a fusion protein comprising a member of the pro-apoptotic Bcl-2 family, such as cpIL-4-Bad, according to the present invention, wherein the fusion protein is used to neutralize or down-regulate the tumor microenvironment. It may be administered to a subject prior to or during therapy, immunotherapy, or the like.

Non-malignant cells include fibroblasts, myofibroblasts, glial cells, epithelial cells, adipocytes, vascular cells (including blood and lymphatic vascular endothelial cells and pericytes), resident and / or recruited inflammation Sexual and immune (eg, macrophages, dendritic cells, bone marrow suppressor cells, granulocytes, lymphocytes, etc.), resident and / or replenishing cells capable of giving rise to or differentiating any of the above non-malignant cells As well as functionally distinct subtypes of the cells as known in the art.

A “subject” is a human, or a mammal, such as a veterinary patient (eg, a rodent such as a mouse or rat, a cat, a dog, a cow, a horse, a sheep, a goat, or other livestock) in need of treatment. Can be In some embodiments, a “subject” may be a clinical patient, clinical trial volunteer, experimental animal, and the like. The subject may have or be suspected of having or being at risk of having a condition characterized by cell proliferation, as described herein, or be diagnosed with a condition characterized by cell proliferation. Or a control subject that has been confirmed not to have the condition characterized by cell proliferation. Diagnostic methods for conditions characterized by cell proliferation, and clinical depictions of such diagnoses, are known to those of skill in the art.

The composition can be a solution, suspension, emulsion, sustained release formulation, or powder, and can be formulated with a pharmaceutically acceptable carrier. The composition can be formulated as a suppository, with traditional carriers and binders such as triglycerides. The term "pharmaceutically acceptable carrier" refers to a carrier medium or vehicle that does not interfere with the effectiveness of the biological activity of the active ingredient and is not toxic to the host or subject.

The fusion protein can be delivered together with a pharmaceutically acceptable vehicle. In one example, the vehicle can enhance stability and / or delivery properties. Thus, the present disclosure also includes artificial membrane vesicles (including liposomes, noisomes, nanosomes, etc.)
A suitable vehicle, such as microparticles or microcapsules, is provided in the formulation of the fusion protein or provided as a colloidal formulation comprising a pharmaceutically acceptable polymer. Such media /
The use of a polymer can be beneficial in achieving sustained release of the sustained release fusion protein. Alternatively, or in addition, the fusion protein formulation may include additives that stabilize the protein in vivo, such as human serum albumin, or other stabilizers for protein therapeutics known in the art. The fusion protein formulation can also include one or more viscosity enhancing agents that act to prevent reflux of the formulation, for example, when administered by injection or via a catheter. Such viscosity enhancers include, but are not limited to, biocompatible glycols and sucrose.

Pharmaceutical compositions containing one or more fusion proteins can be prepared by methods known in the art.
It may be formulated as a sterile injectable aqueous or oleaginous suspension using one or more suitable dispersing or wetting agents and / or suspending agents as described above. The sterile injectable preparation may be a sterile injectable injection or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable media and solvents that can be employed include, but are not limited to, water, Ringer's solution, Ringer's lactate solution, and isotonic sodium chloride solution. Other examples include sterile, fixed oils which are conventionally employed as a solvent or suspending medium, and various brands of fixed oils, including, for example, synthetic mono- or diglycerides. Fatty acids such as oleic acid can also be used in preparing injectables.

In some embodiments, the fusion protein is conjugated to a water-soluble polymer, for example, to increase stability or circulating half-life, or reduce immunogenicity. Clinically acceptable, water-soluble polymers include polyethylene glycol (PEG), polyethylene glycol propionaldehyde, carboxymethyl cellulose, dextran, polyvinyl alcohol (PV
A), polyvinylpyrrolidone (PVP), polypropylene glycol homopolymer (PP
G), including, but not limited to, polyoxyethylated polyols (POGs) (eg, glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylated glucose, and other carbohydrate polymers. Not limited. Methods for conjugating polypeptides to water-soluble polymers such as PEG are described, for example, in US Patent Publication No. 20050106148 and references therein. In one example, the polymer is a pH-sensitive polymer designed to enhance release of the drug from the acidic endosomal compartment into the cytoplasm (see, for example, Henry et ah, Biomacromolecules 7 (8): 2407-14, 2006). .

Typically, vaccines are prepared in injectable forms, either as solutions or suspensions. Solid forms suitable for injection can also be prepared as emulsions or using the polypeptides encapsulated in liposomes. The cells are injected in any suitable carrier known in the art. Suitable carriers typically include macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactive virus particles. . Such carriers are well-known to those skilled in the art. These carriers can also function as adjuvants.

Adjuvants are immunostimulants that enhance the effectiveness of the vaccine. Effective adjuvants include aluminum salts such as aluminum hydroxide and aluminum phosphate, muramyl peptides, bacterial cell wall components, saponin adjuvants, and other substances that act as immunostimulants to enhance the effectiveness of the composition. But not limited to these.

The vaccine is administered in a manner compatible with the dosage formulation. An effective amount refers to a vaccine administered in a single dose, multiple dose schedule, that is effective for treating or preventing a disease or disorder. Preferably, such doses are effective to inhibit neoplastic growth. The dose to be administered will vary depending on the subject to be treated, the subject's health and physical condition, the potential of the subject's immune system to produce antibodies, the degree of protection desired, and other relevant factors. The exact amount of active ingredient required will be at the discretion of the practitioner.

The pharmaceutical compositions described herein can contain an effective amount of an agent to achieve its intended purpose.
Includes one or more fusion proteins. Typically, a composition comprising a fusion protein containing a pro-apoptotic Bcl-2 family member already has a disease, disorder or condition characterized by cell proliferation, or has such a disease, disorder or condition. A patient at risk, such as a condition, is administered in an amount sufficient to cure or at least partially arrest the symptoms associated with cell proliferation, or to reduce cell growth.

Those skilled in the art will therefore recognize that the dosage to be administered is not subject to the defined limitations. Prior to administration for therapeutic purposes, the dosage of the fusion protein may need to be adjusted or modified for a particular purpose, e.g., the concentration of the fusion protein required for systemic administration may be It may differ from that used for administration. Similarly, the toxicity of a therapeutic agent may vary depending on the mode of administration and the overall composition used (eg, buffers, diluents, additional chemotherapeutic agents, etc.).

An “effective amount” of a pharmaceutical composition according to the present invention includes a therapeutically effective amount or a prophylactically effective amount. "Therapeutically effective amount" refers to an amount of a fusion protein that is effective, at the dosage and for the required period, to ameliorate the symptoms of the disease, disorder or condition to be treated. The therapeutically effective amount of a compound can vary depending on factors such as the condition, age, sex, and weight of the subject, and the ability of the compound to elicit the desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the fusion protein are outweighed by the therapeutically beneficial effects. Determination of the therapeutically effective dose of a compound will be made with the potential of those skilled in the art. For example, a therapeutically effective dose may be initially determined in either a cell culture assay, such as those described herein, or in an animal model.
Can be estimated. A "prophylactically effective amount" is an amount of a fusion protein effective to achieve the desired prophylactic result, such as delaying the onset of symptoms of a neurological disorder or continuing remission of cancer, at the dosage and for the required period of time. Say. Animal models can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in other animals, including humans, using standard methods known to those skilled in the art.

The concentration of the fusion protein in the final formulation can be at least 0.1 mg / mL, such as at least 1 ng / mL or at least 1 μg / mL or at least 1 mg / mL. For example, the concentration in the final formulation is about 0.01 μg / mL and about 1,000 μg
/ ML. In one example, the concentration in the final formulation is about 0.01 m
g / mL and about 100 mg / mL.

In some embodiments, the fusion protein comprising the anti-apoptotic Bcl-2 family protein, or a fragment thereof, is at a concentration ranging from about 10 ng / mL to about 10,000 ng / mL, or about 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng
/ ML, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL,
350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL, 550 n
g / mL, 600 ng / mL, 650 ng / mL, 700 ng / mL, 750 ng / mL
, 800 ng / mL, 850 ng / mL, 900 ng / mL, 950 ng / mL, 100
0 ng / mL, 1500 ng / mL, 2000 ng / mL, 2500 ng / mL, 300
0 ng / mL, 3500 ng / mL, 4000 ng / mL, 4500 ng / mL, 500
0 ng / mL, 5500 ng / mL, 6000 ng / mL, 6500 ng / mL, 700
0 ng / mL, 7500 ng / mL, 8000 ng / mL, 8500 ng / mL, 900
It is administered at any value in between, such as 0 ng / mL, 9500 ng / mL, or 10000 ng / mL.

In some embodiments, the fusion protein comprising a pro-apoptotic Bcl-2 family protein, or a fragment thereof, is administered at a concentration ranging from about 0.1 ng / mL to about 10,000 ng / mL.

However, the actual amount of the compound (s) to be administered will depend on the condition to be treated, the route of administration chosen, the compound actually administered, the age, weight and responsiveness of the individual patient, and It will be appreciated that the decision will be made by the physician in view of the relevant circumstances, including the severity of the patient's symptoms. The above dosage ranges are given by way of example only and are not intended to limit the scope of the invention in any way. In some cases, dosage levels below the lower end of the range may be too sufficient, while in other cases, higher doses may be used without adverse side effects, such as, for example, a daily dose. For global administration, it may be done first by dividing the larger dose into several smaller volumes.

Those skilled in the art will appreciate that the dosage will depend, inter alia, on the type of fusion protein used and the type of disorder or condition being treated.

In general, fusion proteins according to the present disclosure substantially comprise human sequences and are therefore less antigenic than other molecules or immunotoxins, including, for example, non-human sequences. In some embodiments, a fusion protein according to the present disclosure has at least 80%, for example, 80%, 85%,
Contains 90%, 95%, 96%, 97%, 98%, 99% or 100% human sequences. In some embodiments, a fusion protein according to the present disclosure may be, for example, an immunotoxin, or an IL-
It can be administered at substantially lower doses than a native IL-4R binding protein such as IL-4 or IL-13.

In some embodiments, the fusion protein may elicit a certain level of antibody response when administered to a subject, which in some cases may lead to undesirable side effects.
Thus, if necessary, the antigenicity of the fusion protein can be assessed as known in the art and / or as described herein. For example, the in vivo toxic effects of the fusion proteins can be assessed by measuring their effect on animal weight during treatment and by performing hematological profiles and liver enzyme analysis after the animal dies. The general toxicity of the fusion protein can be tested by methods known in the art. For example, the overall systemic toxicity of the fusion protein is 100% of mice (ie, LD ₁₀₀ ) or 50% of mice (ie, LD ₅ ) after a single intravenous injection.
₀ ) can be determined by determining the dose at which death occurs. LD ₁₀₀ or LD
A dose of at least about 2, 5, or less than ₅₀ times the ₅₀ can be selected for administration to other mammals, such as humans.

The kinetics and magnitude of the antibody response to the fusion proteins described herein are
It can be determined in immunized mice and they can be used to facilitate the development of regimens that may be used in immunized humans. C57
Intravenous doses of the fusion protein are administered to immunized mice such as the BL6 strain. Mice are sacrificed at various intervals (eg, after a single dose, after multiple doses) to obtain serum. An ELISA-based assay can be used to detect the presence of the anti-fusion protein antibody.

Serum samples from mice can be assessed for the presence of anti-fusion protein antibodies as known in the art. As another example, Stickler et al., J. Immunotherapy, 23: 654-660, 200.
As described in Section 0, epitope mapping can also be used to determine the antigenicity of a protein. Briefly, immune cells known as dendritic cells and CD4 + T cells are isolated from blood from community donors that have not been exposed to the protein of interest. The synthetic small peptide corresponding to the protein length is then added to the cells in culture. Proliferation in response to the presence of a particular peptide suggests that a T cell epitope is included in the sequence. This peptide sequence can then be deleted or modified in the fusion protein, thereby reducing its antigenicity.

Therapeutic efficacy and toxicity can also be determined, for example, by a semi-effective dose, ie, ED ₅₀ (ie, 50% of the population).
And a semi-lethal dose, ie, by determining the LD ₅₀ (ie, the dose lethal to 50% of the population) by standard pharmaceutical means. The dose ratio between therapeutic and toxic effects is known as the “therapeutic index” and is the ratio, LD ₅₀ / E
D can be expressed as _50. The data obtained from cell culture assays and animal studies can be used in formulating a wide range of dosage for human or animal use. The dosage contained in such compositions is typically within a range of concentrations that include the ED ₅₀ and is low or non-toxic. The dosage varies within this range depending upon the dosage form employed, sensitivity of the subject, and the route of administration.

Administration of the fusion protein can be within a lesion, for example, directly into a site of apoptotic tissue; into a site requiring cell growth; the cell, tissue or organ is at risk of cell death. Into the site; or by direct injection into the site of hyperproliferation or into the tumor. Alternatively, the fusion protein can be administered systemically. For methods of combination therapy that include administering the fusion protein in combination with a chemotherapeutic agent, the order in which the compositions are administered is interchangeable. Simultaneous administration is also envisioned.

Typically, in the treatment of cancer, the fusion protein will be administered systemically to the patient, for example, by bolus injection or continuous infusion into the patient's bloodstream. Alternatively, the fusion protein may be administered locally, at the site of the tumor (within the tumor). When the fusion protein is administered intratumorally, the administration can be by any route, such as, for example, local, local, localized, systemic, convection enhanced delivery, or a combination thereof. .

When used in combination with one or more known chemotherapeutic agents, the compound can be administered before or after administration of the chemotherapeutic agent, or they can be administered simultaneously. One or more chemotherapeutic agents may be administered systemically, for example, by bolus injection or continuous infusion, or they may be administered orally.

For administration to animals, pharmaceutical compositions can be formulated for administration by various routes. For example, the compositions can be formulated for topical, rectal or parenteral administration, or for administration by inhalation or spray. As used herein, the term "parenteral" refers to subcutaneous injection, intravenous,
Includes intramuscular, intrathecal, intrasternal injection or infusion techniques. Direct injection or infusion into the tumor is also contemplated. Convective enhanced delivery can also be used to administer the fusion protein.

In one example, a fusion protein can be injected into a subject with cancer using an administration approach similar to multiple injection approaches in brachytherapy. For example, multiple aliquots of a purified fusion protein, in the form of a pharmaceutical composition or formulation, and made into a suitable dosage unit, may be injected with a needle. Methods that can be employed in addition to administering the fusion protein will be apparent to those skilled in the art. Such methods include, for example, using a catheter or implantable pump to provide continuous infusion of the fusion protein to a subject in need of treatment.

A software planning program, such as is known in the art, for treating brain tumors or other localized tumors, e.g., for placement of a fusion protein infusion catheter,
It can be used in combination with brachytherapy procedures and ultrasound. For example, positioning and placement of the needle can be accomplished generally under ultrasound guidance. The total amount, and thus the number of injections and deposits administered to the patient, can be adjusted, for example, according to the volume or area of the organ to be treated. An example of a suitable software planning program is the Brachytherapy Treatment Planning Program Variseed 7.1 (Varian Medical Sys- tem) for enhanced convective delivery to the brain.
tems, Palo Alto, Calif.) or iPlan (BrainLab, Munich, Germany). Such an approach has been successfully implemented, especially in the treatment of prostate and brain cancer.

Fusion proteins can be used to inhibit cell survival or inhibit cell proliferation in the central nervous system (CNS). If the site of delivery is the brain, the fusion protein must be able to deliver to the brain. The blood-brain barrier limits the uptake of many therapeutics from the general circulation into the brain and spinal cord. Molecules that cross the blood-brain barrier use two major mechanisms: free diffusion and facilitated transport. Due to the presence of the blood-brain barrier, the use of certain drug delivery strategies may be required to reach beneficial concentrations of certain fusion proteins in the CNS. Delivery of the fusion protein to the CNS can be accomplished by several methods.

One method is based on neurosurgical techniques. For example, the fusion protein can be delivered by direct physical introduction into the CNS, such as by intraventricular, intralesional, or intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. The method of introduction is also provided by a rechargeable or biodegradable device. Another approach is disruption of the blood-brain barrier by substances that increase the permeability of the blood-brain barrier. Examples include intra-arterial infusion of low-diffusible substances such as mannitol, drugs that enhance cerebral vascular permeability such as etoposide, or vasoactive drugs such as leukotriene, or enhanced convection delivery (CED) by catheter. In addition, it may be desirable to administer the composition locally to the area in need of treatment, for example, by local injection during surgery, by injection, by use of a catheter, or by implantation. ) (The implant may be of a porous, non-porous or gel-like material, including membranes such as silastic membranes, or fibers). A preferred membrane is Gliadel® (Eisai Inc.).

Non-viral approaches can be taken for introducing therapeutic agents into cells in need of modulation of cell death (eg, cells of a patient). For example, lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al.,
Neuroscience Letters 17: 259, 1990; Brigham et al., Am. J. Med.Sci. 298: 278, 198
9; Staubinger et al., Methods in Enzymology 101: 512, 1983), asialoorosomucoid-polylysine junction (Wu et al., Journal of Biological Chemistry 263: 14621, 19).
88; Wu et al., Journal of Biological Chemistry 264: 16985, 1989) by administering nucleic acid molecules into cells or by microinjection under surgical conditions (Wolff et al., Science). 247: 1465, 1990), the nucleic acid molecule can be introduced. Preferably, the nucleic acids are administered in combination with liposomes and protamine.

Gene transfer can also be accomplished using viral means, including in vitro transfection. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into cells. Transplantation of the fusion protein into a patient's diseased tissue involves introducing a normal nucleic acid ex vivo into a culturable cell type (eg, an autologous or xenogeneic primary or progeny cell) and then transferring the cell (or progeny thereof). It can also be achieved by injection into the targeted tissue.

CDNA expression for use in polynucleotide therapy can be achieved by using a suitable promoter (
For example, human cytomegalovirus (CMV), Simian virus 40 (SV40),
Or metallothionein promoter) and can be controlled by any of the appropriate mammalian control elements. For example, if desired, enhancers known to preferentially direct gene expression in certain cell types can be used to direct nucleic acid expression. The enhancers used can include, but are not limited to, those characterized as tissue-specific or cell-specific enhancers. Alternatively, if the genomic clone is to be used as a therapeutic construct, control is provided by cognate control sequences, or, if desired, by control sequences from a heterologous source, including any of the above promoters or control elements. Can be mediated.

  The present invention is further described in the following examples.

  Example 1

  This example describes the generation of a circularly permuted IL-4 protein.

The coding sequence for IL-4 is designed to create and recognize a new amino terminus and a new carboxy terminus. The site of reorganization is selected and the coding region is created synthetically or using the native sequence as a template. PCR can be used to amplify separate coding regions, and the design of the 5 'and 3' ends of such separate fragments to overlap allows for the formation of new coding sequences, where A newly prepared peptide, for example, which can be the 40th amino acid in a native protein, can encode the first amino acid. Specific examples of making cyclic permuted ligands are provided in US Pat. No. 6,011,002.

  The following sequence is used as a reference.

Example 2

cpIL-4-BAD was prepared using standard techniques, and cpIL4-PE was used for cancer cells expressing IL-4Rα in vitro and for IL-4Rα-positive xenograft tumors in an athymic nude mouse model. It was used as a reference. As a result, cpIL-4
-BAD was suggested to be effective in killing cancer cells and regressing tumors.

More specifically, a cpIL-4-BAD fusion protein was prepared by transducing BL21 (DE3) pLysS bacteria with the chimeric plasmid cpIL-4BAD in the pET24a expression vector (FIG. 1). LB containing kanamycin with newly transformed bacteria
Seeded on top. Colonies were picked, transferred to 1 liter of superbroth, enriched in glucose, MgCl ₂ and kanamycin, and cells were induced with 1 mM IPTG. Cell growth was monitored for 6 hours. The IL-4-BAD fusion protein is HiTrap C
Refolding and purification using olumn and then two MonoS columns, then the cpIL-4-BAD fraction was evaluated by SDS-PAGE electrophoresis. In this strategy,
About 0.980 mg / L cpIL-4BAD (pH 6.5), and about 2.930 mg / L
Of cpIL-4BAD (pH 6.8) was obtained.

In quadruplicate in 6-well petri dishes, IL-4Rα positive tumor cells (U251 cells and Da
udi cells) were seeded overnight in complete medium. 0.1 to 1000 ng / mL of cpIL-4
BAD was added and plates were incubated for 5 days at 37 ° C. in a CO ₂ incubator. On day 5, the plates were washed with 1 × PBS and cells were detached by trypsinization. After inactivation of trypsin, viable cells were counted using trypan blue dye exclusion technique.

As a result, the cpIL-4BAD fusion protein was converted to U 25 in vitro in a concentration-dependent manner.
One cell was killed, suggesting an IC ₅₀ value of about 0.6 ng / mL (FIG. 2).
A 100-fold excess of IL-4 blocked the ability of cpIL-4BAD to kill U251 cells, demonstrating that cpIL-4BAD activity was specific (FIG. 3). In a 5-day cell viability assay, the IC ₅₀ value was about 0.3 ng / mL to 1000 ng.
/ ML.

The cpIL-4BAD fusion protein killed Daudi cells in vitro in a concentration-dependent manner, with an IC ₅₀ value of about 0.1 ng / mL (FIG. 4). A 100-fold excess of IL-4 also blocks cpIL-4BAD's ability to kill Daudi cells, and cpIL-4B
It demonstrated that the AD activity was specific (FIG. 5). In a 5-day cell viability assay, the IC ₅₀ value increased from about 0.3 ng / mL to over 1000 ng / mL.

In a colony formation assay, the cpIL-4BAD fusion protein was also found to reduce the number of colonies of IL-4Rα positive tumor cells. More specifically, 5000 U 25
One cell was seeded on a 10 cm culture plate (six plates in total), cpIL-4BAD was added, and the cells were incubated for 10 days. Colonies were stained with Crystal Blue and counted. As a result, it was suggested that cpIL-4BAD reduced the number of U251 colonies in vitro in a concentration-dependent manner, and the IC ₅₀ value was about 5 ng / mL (FIG. 6).

In athymic mice after developing subcutaneous glioma tumors with U251 tumor cells, cp
The efficacy of the IL-4BAD fusion protein was assessed. More specifically, cpIL-4BA
D was injected intratumorally (IT) at 100 μg / kg (6 injections) and intraperitoneally (IP) at 100 μg / kg.
kg (6 injections). 0.2% HAS / PBS was used as a vehicle control. The results assessed were tumor size and survival. The results indicated that intratumoral administration of cpIL-4BAD significantly regressed tumor growth after 6 injections compared to animals treated with placebo control. Intraperitoneal administration dramatically regressed tumors compared to intratumorally treated mice.
Tumors in both groups of mice recurred beyond day 40, where mice in the control group were euthanized on day 36 for ethical reasons as they had a tumor of 2000 mm3 (FIG. 7). cpI
Mice treated with L-4BAD also survived longer than placebo-treated mice (FIG. 8).

  Example 3

Six more IL-4Rα positive cell lines were identified as cpIL-4BAD fusion protein and cp
Their sensitivity to IL-4PE was examined under similar experimental conditions. IC ₅₀ values were determined by counting cells using the trypan blue dye exclusion technique (Table 3).

  Example 4

The IL-4BAD, cpIL-4BAD and cpS4-BAD fusion proteins were expressed and purified. More specifically, IL-4BAD, cpIL-4BAD and cpS4-BAD
Was cloned by PCR cloning into the BamFII / XhoI site of the pGW07 E. coli expression vector (FIG. 9). The resulting vector was confirmed by DNA sequencing (FIG. 10).
AD).

Protein expression was performed in E. coli cells. IL-4BAD, cpIL-4BA
D and cpS4-BAD proteins were expressed in insoluble form in 1 L cultures and IMA
Purified under denaturing conditions using C followed by "quick dilution" protein refolding. As determined by non-reducing SDS-PAGE, refolding by "quick dilution" produced intact proteins without aggregates. The final sample size and concentration were as follows: IL-4BAD has 0.2 as measured by UV 280 nm.
Approximately 3.5 mL at 4 mg / mL (UV 280 nm absorbance at 1 mg / mL = 1.14)
Met. cpIL-4BAD was approximately 3.5 mL at 0.23 mg / mL (UV280 nm absorbance at 1 mg / ml = 1.13) as measured by UV280 nm. cpS4
-BAD is about 3.5 mL (1 mg) at 0.23 mg / mL as measured by UV 280 nm.
UV absorbance at 280 nm / ml = 1.25). Proteins were stored in a storage buffer composition: 500 nM NaCL, 10 mM Na-phosphate, pH 7.0, 1% glycerol, 1 μΜ EDTA, 0.01% Tween20.

BL21 (DE3) pLysS-RARE2 cells were transformed with IL-4BAD, cpIL-4B
Transformed with AD and cpS4-BAD protein expression construct, 100 μg / mL A
The cells were seeded on an LB plate supplemented with mp, and incubated at 37 ° C. overnight. The next day, colonies from the plate were scraped and resuspended in LB liquid medium containing 100 μg / mL Amp. The culture was then grown at 37 ° C. with aeration and protein expression was induced with 1 mM IPTG when the OD _{600 of the} cell culture reached approximately 0.5. Induction continued for about 4 hours at 30 ° C. The cell pellet was then collected and stored at -20C. A 10 μL sample of uninduced and induced cultures was dissolved in 50 μL reduced protein loading buffer by boiling at 95 ° C. for 10 minutes and run on an SDS-PAGE gel. Cells from 1 mL samples collected 4 hours after induction were lysed in hypotonic buffer, sonicated and centrifuged at 13,000 rpm for 10 minutes. Aliquots from the soluble and insoluble fractions were boiled in reduced protein loading buffer and SDS-PA
Analyzed on GE gel. The estimated expression level observed for IL-4BAD, cpIL-4BAD protein was greater than 50 mg / L of the crude material. The estimated expression level observed for cpS4-BAD was greater than 50 mg / L. IL-4BAD was found to be almost completely insoluble, and contained some soluble form (less than 5%). cpS4-
BAD was mainly in the insoluble fraction.

The cell pellet from the induced culture was lysed at room temperature, and the inclusion body fraction was collected and PBS-
Washed with T. The insoluble material was solubilized with 8M urea and bound to 3 mL of Ni-charged resin. The resin was washed with 15 column volumes of wash buffer and the bound proteins were eluted with a step gradient elution of 8 column volumes of wash buffer containing imidazole. 7.5 μL from each fraction was analyzed on an SDS-PAGE gel. The maximum amount of IL-4BAD (about 6
mg) and cpIL-4BAD (about 8 mg) were combined and refolded. The remaining fraction was stored at -20C.

The IL-4BAD and cpIL-4BAD fractions were combined, reduced by the addition of 1 mM DTT, and supplemented with urea at increasing concentrations with each dialysis buffer change (150 mM N2).
aCl, 10 mM HEPES, pH 7.4, 0.01% Tween 20). After each dialysis step, the protein concentration was determined by UV spectroscopy: IL-4BAD was about 0.8 mg; cpIL-4BAD was about 0.45 mg. The sample was then run on an SDS-PAGE gel.

25 μL of each protein (〜0.6 mg / mL, 200 mM imidazole fraction) was diluted in 1 mL of the following buffer: 20 mM HEPES, pH 7.4, 1% glycerol,
10 μΜ EDTA, 0.01% Tween (buffer 1); 10 mM Na-phosphate, p
H7.0, 1% glycerol, 10 μΜ EDTA, 0.01% Tween (buffer 2)
And PBS, pH 7.2 (buffer 3). Samples were stored overnight at room temperature. The next day, sample 1
After spinning at 3,000 rpm for 10 minutes, the presence of pellets in each sample was observed and recorded in Table 2. (+++) indicates a large amount of pellets, (-) indicates no visible pellets.

The samples were also analyzed on a non-reducing SDS-PAGE gel. The refolded samples were concentrated to 100 μL using Amicon 10 kDa MWCO, spun down, and 7.5 μL aliquots of each sample were run on an SDS-PAGE gel.

The purification process was repeated using rapid dilution folding. IL-4BAD, cp
4 ml of a 200 mM imidazole fraction containing IL-4BAD or cpS4-BAD,
200 mL of refolding buffer (500 mM NaCl, 10 mM Na-phosphate, pH 7.0 or 6.0 or 7.8, 1% glycerol, 10 μΜ EDTA, 0.0
(1% Tween), incubate overnight at room temperature, 4,000 rpm
m, spin down at 4 ° C. for 20 minutes, concentrate to 3 mL using Amicon 10 kDa MWCO, and buffer using a DG-10 column with storage buffer (500 mM NaCl, 10 mM NaCl).
-Phosphate, pH 7.0 or 6.0 or 7.8, 1% glycerol, 1 μΜ EDTA,
(0.01% Tween). The final sample concentrations were as follows: about 0.
3.5 mL of IL-4BAD at 24 mg / mL; 3.5 mL at about 0.23 mg / mL
CpIL-4BAD. The final sample was run on an SDS-PAGE gel.

The final concentration of cpS4-BAD was about 3.5 mL at about 0.23 mg / mL. The final sample was run on an SDS-PAGE gel.

  Example 5

The pKFR4-BAD-H6 fusion protein (FIGS. 11A-B) was prepared as follows. The cDNA of pKFR4-BAD-H6 was obtained from Ndel / PET-21a (+) vector.
PCR cloning was performed at the Xhol site. The vector was then transformed into HMS174 (DE3) cells and induced with 0.1 mM IPTG for 3 hours. SDS- before and after induction
Run on a PAGE gel. Pellet the cells and add 20mM Tris-HCl, 300m
Dissolution was achieved by sonication in a buffer of pH 8.0 containing M NaCl, 20 mM imidazole, and the samples were run on SDS-PAGE gels.

The inclusion bodies were dissolved in a lysis buffer of 20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 20 mM β-ME, 7M GuaHCl, pH 8.0.
Thereafter, the supernatant was purified by Ni ²⁺ affinity chromatography. pKFR4
-BAD-H6 contains 20 mM Tris-HCl, 300 mM N under reducing and non-reducing conditions.
Eluted with aCl, 300 mM imidazole, 8 M urea, pH 8.0. Samples were taken throughout the purification process.

After purification, pKFR4-BAD-H6 was dialyzed in dialysis buffer: 0.1% TFA, 30% acetonitrile under reducing and non-reducing conditions to give a concentration of about 2.67 mg / mL.

  Example 6

To evaluate the efficacy of the fusion protein on cell lines expressing IL-4R, 1
0% FBS (from Life Technologies), 2 mM L-glutamine (from Gibco) and 10%
Type I IL in RPMI 1640 (from Gibco) containing mM HEPES (from Gibco)
HH suspension cells expressing -4R were cultured. cpS4-BAD was added to the cell suspension. The cell suspension was poured into 96-well Isoplates at a volume of about 1 × 10 ⁴ cells / well. The cell suspension was incubated for 48 hours at 37 ° C. in a 5% CO ₂ incubator. Before the end of the 48 hour incubation, complete media containing 100 [mu] Ci / mL [< ³ > H] -thymidine was prepared and 10 [mu] L was added to each well of the culture. After 6 hours incubation with [< ³ > H] -thymidine, 50 [mu] L of 50% trichloroacetic acid was slowly added to each well and incubated at 4 [deg.] C for 2 hours. It washed and air-dried and then plate with dH 2 ₀ 5 times. 100 μL of scintillation fluid was added to each well and the plate was left at room temperature overnight. The next day, radioactivity was read on a MicroBeta Trilux. Data was collected and normalized using readings from control wells (cells only). The background reading (blank) was subtracted from all well readings and the inhibition (% inhibition) was calculated. The IC _{50 of} cpS4-BAD was about 126.3 ng / mL, which was about 3-fold higher than MDNA55 (cpIL4-PE; 362.4 ng / mL) used as a reference.

  All references are incorporated herein by reference.

The present invention has been described with respect to one or more embodiments. However, it will be apparent to one skilled in the art that certain changes and modifications may be made without departing from the scope of the invention as defined in the claims.

Claims (29)

Interleukin-4 (IL-4) receptor binding protein and pro-apoptotic Bc
A fusion protein comprising a 1-2 family polypeptide. The fusion protein of claim 1, wherein the IL-4 receptor binding protein is cyclically permuted (cp). 3. The fusion protein according to claim 1, wherein the pro-apoptotic Bcl-2 family polypeptide comprises a BH3 domain. The pro-apoptotic Bcl-2 family polypeptide comprising a BH3 domain,
The fusion protein according to claim 3, which is Bad, Bik / Nbk, Bid, Bim / Bod, Hrk, Bak or Bax. The pro-apoptotic Bcl-2 family polypeptide comprising a BH3 domain,
The fusion protein according to claim 3 or 4, further comprising a mutation that reduces phosphorylation. The fusion protein according to claim 5, wherein the pro-apoptotic Bcl-2 family polypeptide comprising the BH3 domain further comprising a mutation that reduces phosphorylation is a Bad polypeptide. Claims: The fusion protein is capable of inhibiting cell survival, inhibiting cell proliferation, or enhancing cell death or apoptosis of a target cell expressing the IL-4 receptor (IL-4R). The fusion protein according to any one of claims 1 to 6. The IL-4 receptor binding protein is a mutant IL-4 or IL-13 that is selective for binding to a type I or type II IL-4R. A fusion protein as described. The variant IL-4 selective for binding to type II IL-4R comprises a KFR variant or a KF variant, or the variant selective for binding to type I IL-4R IL-
9. The fusion protein of claim 8, wherein 4 comprises an RGA variant. The fusion protein according to claim 8, wherein the mutant IL-13 comprises an A11 mutant or a DN mutant.   The fusion protein according to any one of claims 1 to 10, further comprising a linker. The fusion protein according to claim 11, wherein the linker has the sequence GS or is a ubiquitin or ubiquitin variant molecule. 3. The fusion protein according to claim 1, comprising the amino acid sequence of any one of SEQ ID NOs: 24 to 27. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 13.   A nucleic acid molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: 35-38.   A vector comprising the nucleic acid molecule according to claim 14.   A host cell comprising the vector according to claim 16. 14. The fusion protein according to any one of claims 1 to 13, 14 or 1
A pharmaceutical composition comprising the nucleic acid molecule of claim 5, the vector of claim 16, or the host cell of claim 17. 14. The fusion protein according to any one of claims 1 to 13, 14 or 1
A method for inducing cell death, comprising administering the nucleic acid molecule of claim 5, the vector of claim 16, or the host cell of claim 17, to a subject in need thereof. Contacting a target cell expressing IL-4R with a fusion protein according to any one of claims 1 to 13, a nucleic acid molecule according to claim 14 or 15, or a vector according to claim 16. A method for inducing cell death, comprising: 14. The fusion protein according to any one of claims 1 to 13, 14 or 1
18. A method for treating cancer, comprising administering the nucleic acid molecule according to 5, the vector according to claim 16, or the host cell according to claim 17 to a subject in need thereof. Contacting a neoplastic cell that expresses IL-4R with a fusion protein according to any one of claims 1 to 13, a nucleic acid molecule according to claim 14 or 15, or a vector according to claim 16. A method of treating cancer, comprising: A method of treating cancer, comprising administering IL-4 to a tumor microenvironment in a subject in need thereof.
A non-malignant cell that expresses R can be a fusion protein according to any one of claims 1 to 13, a nucleic acid molecule according to claim 14 or 15, a vector according to claim 16, or a vector according to claim 17. A method comprising contacting the described host cell. 24. The method of claim 23, wherein the non-malignant cells are contacted before the subject starts treatment. A method for treating a hyperproliferative disorder or a differentiation disorder, comprising: a fusion protein according to any one of claims 1 to 13; a nucleic acid molecule according to claim 14 or 15;
18. A method comprising administering the vector according to the above or the host cell according to the above 17 to a subject in need thereof. 26. The method of claim 25, wherein said hyperproliferative or differentiation disorder is fibrosis or hyperplasia, an inflammatory condition or an autoimmune condition. The fibrosis or hyperplasia is pulmonary fibrosis or hyperplasia (such as benign prostatic hyperplasia), cardiac fibrosis, or hepatic fibrosis, and the inflammatory condition is prostatitis, spring keratoconjunctivitis, atherosclerosis, 27. The method of claim 26, wherein the disease is idiopathic pneumonia, and the autoimmune condition is Graves' disease. 14. A method according to any one of claims 1 to 13 in a subject in need thereof for inducing cell death or for treating cancer or for treating a hyperproliferative or differentiation disorder. Use of a fusion protein, a nucleic acid molecule according to claim 14 or 15, a vector according to claim 16, or a host cell according to claim 17. The method according to any one of claims 19, 21, 23 to 27, wherein the subject is a human.
Or the use according to claim 28.

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